268 J. D. SMITH 



and their percentage dissociation. This force is opposed by that due to the 

 resistance of flow of the molecule through the liquid, which is proportional 

 to the velocity of motion of the molecule (v) and equal to (Kv) , where K is a 

 function of the size and shape of the molecule and the retarding effects of 

 other ions in the solution. The molecules thus migrate with a constant 

 velocity equal to (EQ/K) . 



Using the dissociation constants of the ionizable groups in nucleic acid 

 components and making certain approximations as to the nature of the 

 resistance to flow of the charged molecules through the fluid, the relative 

 mobilities of these substances may be calculated. This is best illustrated in 

 the case of nucleotides and polynucleotides. 



2. Separation of Nucleotides and Polynucleotides 



The ionizing groups in nucleotides and polynucleotides are the primary 

 and secondary phosphate groups, the amino groups of adenine, guanine, 

 and cytosine and its derivatives, and the enol groups of guanine, cytosine, 

 uracil, and thymine. Table I gives the pK values of these groups for a num- 

 ber of nucleotides. [For a more complete discussion, compare Jordan, Chap- 

 ter 13.] The dissociation constants of the pairs of isomeric ribonucleoside-2'- 

 and -3 '-phosphates are very close to each other, so that all calculations 

 have been made with the values of Levene and Simms^'^ which are probably 

 based on mixtures of the two isomers. The dissociation constants of the 

 deoxy ribonucleotides have not been determined, but to a close approx- 

 imation they may be assumed to be identical with those of the correspond- 

 ing ribonucleotides. In Figure 1 are plotted the dissociation curves for the 

 ionizing groups of the four nucleotides, adenylic, guanylic, cytidylic, and 

 uridylic acids. 



The enol groups of guanylic, cytidylic, and uridylic acids having pK 

 values of 9.5 or above are not dissociated at the pH values useful for most 

 separations, and so for the moment may be ignored, but the primary and 

 secondary phosphate groups and the amino groups are most important. 

 The pK values of the primary phosphate dissociations which lie between 

 0.7 and 1.0 are too close to each other to be used for the separation of the 

 nucleotides; and also some nucleotides and most polynucleotides are un- 

 stable in this pH range. The separation of adenylic, guanylic, cytidylic, 

 and uridylic acids is possible on the basis of the differences in the pK values 

 of the amino groups, which lie between 2 and 5. In this pH range the pri- 

 mary phosphate groups are completely dissociated and the secondary 

 phosphate groups uncharged. The most suitable pH for the separation is 



» P. A. Levene and H. S. Simms, J. Biol. Chem. 65, 519 (1925). 



* P. A. Levene, L. W. Bass, and H. S. Simms, J. Biol. Chem. 70, 229 (1926). 

 3 P. A. Levene, H. S. Simms, and L. W. Bass, J. Biol. Chem. 70, 243 (1926). 



* P. A. Levene and H. S. Simms, /. Biol. Chem. 70, 327 (1926). 



